Regulation of Innate Immunity and Inflammation Through Nuclear Reprogramming We propose to advance our mechanistic studies of innate immunity and inflammation in severe infections that evolve into their end stage, septic shock, thereby threatening the lives of wounded soldiers on the battlefield, our Veterans, and the civilian population. Those Veterans (and non-Veteran patients) between the ages of 65 to 85 are particularly prone to septic shock with a 45% mortality rate while many survivors suffer debilitating cognitive decline. We are pursuing our long-term goal to decode innate immunity and inflammation with a new class of cell-penetrating Nuclear Transport Modifiers (NTMs) that reprogram the inflammatory regulome. One of these anti-inflammatory peptides achieved an unprecedented enhancement of innate immunity-mediated bacterial clearance in the lungs (700-fold), spleen (300-fold), and blood (200-fold), all taking place before antimicrobial therapy. Concurrently, it improved the blood markers of microvascular endothelial injury, including thrombocytopenia. When combined with anti-microbial therapy, NTM increased the survival rate to 55%, compared to a 30% survival rate in the antibiotic-only group. This NTM peptide, cSN50.1, simultaneously targets two nuclear transport shuttles, importins ?5 and ?1. To unravel the mechanism of innate immunity and inflammation that underlie microbial and metabolic disorders, we designed, produced, and tested two novel mono-selective NTMs: (i) Importin ?5-selective cSN50.1?, and (ii) Importin ?1-selective cSN50.1?. Strikingly, the importin ?5-selective peptide produced an 80% survival rate in endotoxin shock induced by lipopolysaccharide (LPS), one of the most potent virulence factors of Gram-negative bacteria responsible for two thirds of septic shock cases. This new NTM also effectively controlled the Western Diet-induced hypersensitivity to lethal endotoxin shock while preventing liver glycogenolysis, which causes hyperglycemia and hypertriglyceridemia linked to the nuclear transport of Carbohydrate Responsive Elements-Binding Proteins (CHREBPs). In turn, the Imp ?1-selective peptide suppressed lipogenesis mediated by the nuclear transport of the Sterol Regulatory Element-Binding Proteins (SREBPs) that control over 30 genes involved in the production of triglycerides, cholesterol, and fatty acids, a hallmark of metabolic syndrome. We hypothesize that the proposed use of selective NTMs with antimicrobial agent(s) would untangle multiorgan damage and mortality in severe infections. Furthermore, we posit that the Importin ?5/SRTFs/CHREBPs and Imp ?1/SREBPs pathways are interdependent, thereby predisposing individuals with metabolic syndrome to hyperacute responses to severe infections. Hence, in Aim 1, we will investigate the mechanism of the NTM?s beneficial action in polymicrobial peritonitis (i.e. the striking enhancement of the innate immunity-mediated clearance of bacteria in the lungs, spleen, kidneys, and blood prior to antimicrobial therapy) by suppressing either Imp ?5/SRTFs/ChREBPs or Imp ?1/SREBPs transport.
In Aim 2, we will analyze the mechanism of the survival gain through the timed and selective inhibition of Imp ?5/SRTFs/ChREBPs or Imp ?1/SREBPs transport without and with antimicrobial therapy. The new selective NTMs will significantly deepen our mechanistic understanding of innate immunity reprogramming using genomic and immunometabolic approaches. Thus, the new pathway-selective NTMs offer us a unique opportunity to (i) decode the transcriptional mechanism of septic shock that integrates microbial and metabolic inflammation and (ii) to develop clinically relevant strategies to reduce the incidence of septic shock, increase survival, and reduce long-term cognitive decline in survivors.

Public Health Relevance

Regulation of Innate Immunity and Inflammation Through Nuclear Reprogramming This project is focused on the mechanistic analysis of the unprecedented enhancement of innate immunity- mediated clearance of bacteria in the lungs, spleen, and blood by a new class of cell-penetrating peptides. These anti-inflammatory peptides selectively target proinflammatory and metabolic nuclear transport pathways. We will also decode both the mechanism of the inflammatory and metabolic injury to multiple organs and increased survival in septic shock. We anticipate that this project will unravel the new genomic and immunometabolic mechanisms of this novel intracellular immunotherapy intended to be combined with antimicrobials. Thus, our innovative anti-inflammatory peptides, coupled with antimicrobials, will pave the way for the improved treatment of severe infections evolving into septic shock in wounded soldiers, Veterans, and the civilian population, thereby reducing the incidence, mortality, and post-septic shock sequelae (such as cognitive decline) in survivors.

National Institute of Health (NIH)
Veterans Affairs (VA)
Non-HHS Research Projects (I01)
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Special Emphasis Panel (ZRD1)
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Veterans Health Administration
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Veach, Ruth Ann; Liu, Yan; Zienkiewicz, Jozef et al. (2017) Survival, bacterial clearance and thrombocytopenia are improved in polymicrobial sepsis by targeting nuclear transport shuttles. PLoS One 12:e0179468
Wylezinski, Lukasz S; Hawiger, Jacek (2016) Interleukin 2 Activates Brain Microvascular Endothelial Cells Resulting in Destabilization of Adherens Junctions. J Biol Chem 291:22913-22923
Wynn, James Lawrence; Wilson, Chris S; Hawiger, Jacek et al. (2016) Targeting IL-17A attenuates neonatal sepsis mortality induced by IL-18. Proc Natl Acad Sci U S A 113:E2627-35